JP2009096805A - Treatment of scarring and related condition using ppar-gamma activator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective medicine for the treatment of a condition affecting the skin. <P>SOLUTION: Use of an activator of the peroxisome proliferator activator receptor gamma PPAR (gamma) in manufacture of a medicament for the treatment of a condition affecting the skin characterized by disordered fibroblast or myofibroblast function, excessive matrix production, nodular fasciitis or Dupuytren's Contracture. The activator includes pioglitazone, thiazolidinedione, 4-phenyloxy-ethyl-5-methyl-2-phenyloxazole, 15-deoxy-δ-12,14-prostaglandin J2, etc. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

FIELD OF THE INVENTION The present invention relates to a novel use (use) for compounds that are effective as agonists at peroxime proliferator activator receptor gamma (PPARγ).

BACKGROUND OF THE INVENTION Excessive scar formation after trauma or surgery can lead to impaired body aesthetics, itching and pain and organ dysfunction. Hypertrophic scars and keloids are skin fibrosis conditions that can occur even with minor injuries, such as ear piercing. These are unique to humans and are characterized by excessive deposition of collagen in the skin and subcutaneous tissue. Keloid sizes range from a few millimeters in the diameter of papules to large or larger footballs. Differences between keloids, hypertrophic scars and normal scars include appearance, histological morphology and cellular functions in response to growth factors.

  Hypertrophic scarring is characterized by elevated fibrous connective tissue in the dermis and adjacent subcutaneous tissue after trauma or burn healing. Keloid is a nodular, often lobed, stiff, mobile non-capsule of a unique hyperplastic fibrous connective tissue that consists of dense collagenous material found in the skin and adjacent subcutaneous tissue after skin injury It typically contains nodes consisting of lumps. Clinically, keloids are defined as scars that grow beyond the normal range of wounds of origin, which, unlike hypertrophic scars, rarely regress over time. About 15-20% of blacks, Hispanics, and Orientals are assessed as having keloids and work is underway to understand possible genetic predispositions.

  Skin fibroblasts and myofibroblasts play a major role in scar formation. In this specification, unless further limited, the term “fibroblast” is understood to include fibroblasts and / or myofibroblasts or all other subtypes of fibroblasts.

  Keloid fibroblasts produce high levels of collagen, fibronectin, elastin, and proteoglycans. Collagen synthesis in keloids is 20 times greater than normal non-scarring skin. It is known that fibroblasts derived from keloids exhibit a four-fold increase in the fibronectin biosynthesis rate. Keloid fibroblasts also show an aberrant response to metabolic modulators such as glucocorticoids, hydrocortisone, growth factors and phorbol esters compared to normal fibroblasts. The altered response of keloid fibroblasts to these metabolic modulators is thought to contribute to the pathogenicity of keloid formation. Fibroblasts from hypertrophic scars also exhibit a mild increase in collagen production in vitro, but their response to metabolic modulators is similar to normal fibroblasts. There is evidence that fibroblasts from hypertrophic scars may exhibit a hyperproliferative phenotype resulting from multiple stimulatory effects present in the wound environment. This phenotype can be reversed when overstimulation, for example, excess growth factors disappear. Keloid fibroblasts, however, represent a unique phenotype that is irreversibly switched on after wound formation. There is also evidence of lipid biology imbalance in keloids.

  After wound formation, the healing mechanism is the same regardless of the cause of injury and can be considered to be in three phases; immediate hemostasis phase, early granulation and reepithelial phase, and late phase of skin repair and remodeling . The hemostatic process is related to the formation of platelet plugs and fibrin clots. Early granulation and reepithelialization phase occurs up to 21 days after injury, depending on the wound site and size. Growth factors derived from platelets stimulate fibroblasts and produce granulated tissue, which includes the growth of epithelial cells leading to re-epithelialization of the collagen matrix and wound surface well supplied by capillaries. The collagen matrix experiences strengthening during the skin repair and remodeling phase and there is a reduction in vascular quality. This phase can last up to 2 years after injury (Martindale, 32nd Edition). Granulated tissue formation usually involves fibroblast replication and mitosis from the tissue to the area of inflammation, and at least a proportion of these regulation towards the myofibroblast phenotype. Angiogenesis (capillary supply) occurs in an equivalent manner, and granulated tissue acquires its typical characteristics. An important part of wound repair is wound contraction or closure. Myofibroblasts (or granulated tissue fibroblasts) are responsible for the development of contractile forces associated with wound contraction and are characterized by the presence of alpha-smooth muscle actin-containing stress fibers. As the wound closes, a gradual progression toward scar tissue occurs, which is related to the loss of vascular cells and myofibroblasts. This phenomenon ends with the establishment of a scar.

  When granulated tissue cells are not excluded, there is the development of pathological scarring, ie hypertrophic and keloid scars, both characterized by a high degree of cytoplasm. It has been shown that the reduction in cell number observed during the transition between granulated tissue and scar is achieved to a large extent by apoptosis. Progressive apoptotic waves are responsible for the progressive disappearance of granulated tissue myofibroblasts. Apoptosis of granulated tissue cells occurs after wound closure and seems to affect the target cells continuously rather than producing a simple wave of cell loss (Gabbiani, Pathol. Res. Pract. 192 (7): 708-711, 1996). Recently, hypertrophic scars have been shown to have a greater number of fibroblast stains (including these as myofibroblasts) for alpha smooth muscle actin than normal skin or healed donor sites . Furthermore, with the time to remodel hypertrophic scars, the number of fibroblasts and myofibroblasts decreases (Nedelec et al, surgery 130 (5): 798-808, 2001).

  In fibroblast cultures derived from keloids, the percentage of apoptotic cells was shown to be 1% for all cell lines. These levels were 50% lower than those found in cell cultures containing normal fibroblasts. Also, fibroblasts derived from the keloid center proliferate more rapidly than those derived from the margin. This suggests that keloids can arise from an abnormal balance between cell growth and cell death (Luo et al, Surgery 107: 87-96, 2001). Other studies show that fibroblasts from keloids have a lower rate of apoptosis and that there was evidence of a mutation in gene p53, an important tumor suppressor gene that links to the apoptotic pathway. (Ladin et al., Wound Repair Regen. 6 (1): 28-37, 1998).

  There is now increasing evidence that pathological scarring conditions can be caused by the persistent presence of a population of myofibroblasts. In addition, there is evidence that dermal myofibroblasts predominate in scleroderma, an unpleasant skin condition, compared to normal skin. Scleroderma is an uncommon fibrotic disorder with high morbidity. It is an invasive disorder where there is excessive immune activation resulting in extensive skin fibrosis due to extensive fibroblast proliferation and endothelial cell dysfunction. It often develops from Raynaud syndrome.

  Treatment strategies including surgery can be effective for hypertrophic scars; however, keloid treatment is poor. Surgery is not a good option because the rate of recurrence without ancillary treatment, such as steroids, varies from 45% to 100%. Treatment of keloids with intralesional steroids such as triamcinolone is often ineffective and the majority of patients have relapses within a year. There are also side effects. There seems to be no way to give prevention in patients who are prone to keloids. There is currently no way to prevent hypertrophic scarring. It is clear that new treatments and preventive measures are necessary for abnormal scarring, especially keloids.

  PPARγ receptors are a subtype of the PPAR (peroxisome proliferator activated receptor) family of nuclear hormone receptors. It has been shown to function as an important regulator in lipid and glucose metabolism, adipocyte differentiation, inflammatory response and energy homeostasis.

  The thiazolidinedione systems rosiglitazone and pioglitazone have been used for the treatment of insulin resistance in type II diabetes. PPARγ thiazolinedione activators have also been shown to have antiproliferative and anti-inflammatory effects on hemangiomyocytes and macrophages. Furthermore, troglitazone has been shown to have an antiproliferative effect on keratinocytes in psoriasis. In this disease, keratinocyte hyperproliferation and immune dysfunction are major components. Such compounds and their usefulness in therapy are described in US-A-5594015, US-A-5824694, US-A-5925657 and US-A-5981586.

  Conversely, activators of the alpha subtype of PPAR (PPARα), including compounds such as clofibrate and gemfibrozil, are described in US-A-6060515 for their ability to enhance epithelial barrier development. It was. Acting via effects on transepithelial water loss, hypertrophic scars and keloids are among the many examples of skin conditions alleged to be susceptible to such treatment.

SUMMARY OF THE INVENTION Surprisingly, it has been shown that PPARγ activators, such as pioglitazone, have the ability to reduce myofibroblast cell numbers. It can therefore be used to treat keloids and hypertrophic scar formation and to treat scleroderma. This prevents the formation of hypertrophic scarring, or accelerates remodeling towards normal scars, where the inhibitory effect of myofibroblasts on selected apoptosis or fibroblast differentiation into myofibroblasts Facilitate or facilitate tissue remodeling of keloids so that it reduces or disappears in size and treats scleroderma symptoms including skin edema, skin thickening and sclerosis, and subsequent atrophy and contracture Based on the understanding that it can.

  The present invention can treat, eg, control or prevent keloids, hypertrophic scars, scleroderma, aberrant myofibroblast function or other skin diseases associated with nodular fasciitis, or Dupuytren's contracture .

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. A: 0 μM, B: 0.1 μM, C: 1 μM, D: 10 μM pioglitazone in fibroblast differentiation medium supplemented with 1 ng / ml TGF-beta1. Arrows indicate examples of DAB positive myofibroblasts.
FIG. 2 A: Fibroblasts (x100) cultured in differentiation medium supplemented with 1 ng / ml TGF-beta1 in the absence of pioglitazone, B: Differentiated fibroblasts cultured with pioglitazone (10 μM) x100), C: high-magnification enlarged view (x200), differentiated fibroblasts showing cells considered to be myofibroblasts based on abundant and expanded cytoskeletal filaments (arrows), D: pioglitazone treatment High magnification (x200) of differentiated (10 μM) differentiated fibroblasts.
FIG. 3 A: Myofibroblasts from cultures not exposed to pioglitazone treatment showing typical size, expanded morphology and predominant myofibrils (arrowheads). B: Pioglitazone-treated culture showing α-smooth muscle positive cells (Oya), clearly contracted and rounded compared to the surrounding normal fibroblasts. There is also evidence of membrane blebbing, suggesting apoptosis rather than necrotic death. C and D: Pioglitazone treated cultures. Myofibroblasts labeled with large arrows show strong but still scattered cytoplasmic staining.

Preferred Embodiments Any PPARγ activator may be used in the present invention. Provided that it has the desired activity. Well known activators of this receptor include the thiazolidinedione series of troglitazone, pioglitazone, rosigglutazone and siglitazone. Other non-thiazolidinedione compounds have recently been identified, for example phenylalkanoic acids are described in WO97 / 31907 and WO00 / 08002, oxazoles and thiazoles are described in WO99 / 38845, and oximinoalkanoic acids are described in WO01 / 38325. Benzoic acid derivatives are described in WO 01/12612, sulfonamides are described in WO 99/58510, β-aryl-α-oxy-substituted alkylated rubonic acids are described in WO 00/50414, and quinolines are described in WO 00/50414. / 64876 and WO00 / 64888. In addition, the natural compound 15-deoxy-Δ-12,14-prostaglandin J2 has also been shown to be a ligand for PPARγ and to have effects mediated via this receptor (Forman et al, Cell 93 (5): 813-819, 1995). Similar effects are also shown for the metabolite of 15-deoxy-Δ-12,14-prostaglandin J2 (Kliewer et al., Cell 83 (5): 813-819, 1995) and for various fatty acids and eicosanoids. (Kliewer et al, PNAS USA 94 (a): 4318-4323, 1997).

  Despite the structural variations allowed by PPARγ, there is substantial similarity in the biological effects for activation of this receptor. PPAR agonists share a common mode of binding to these receptors. Despite differences in the chemical structure of these agonists, the acidic head groups of these agonist ligands accept hydrogen bonds from the tyrosine residues of the AF2 helix and / or the histidine or tyrosine residues of helix-5 (WO01 / 17994). Compounds that have the ability to activate the PPARγ receptor can be expected to be useful in this invention. Preferred agents for this use of the invention include pioglitazone, rosiglitazone, ciglitazone, troglitazone, isaglitazone, darglitazone, and englitazone. It will be appreciated that prodrugs or metabolites of such compounds can be used.

Formulation and Administration For purposes of the present invention, therapeutic compounds are typically administered locally to human patients by intralesional or subcutaneous injection. Oral and parenteral administration are used in appropriate circumstances apparent to the practitioner. Preferably, the composition is administered in a unit dosage form suitable for single administration of the correct dosage.

  To prepare a topical formulation, a therapeutically effective concentration of the compound is placed in a dermatologist vehicle as is known in the art. The amount of therapeutic compound to be administered and the concentration of the compound in the topical formulation will depend on the vehicle selected, the patient's clinical conditions, side effects and the stability of the compound in the formulation. Thus, the physician will use the appropriate preparation containing the appropriate concentration of the therapeutic compound, and select the amount of formulation to be administered depending on the clinical experience of the patient in question or similar patients.

  The concentration of the therapeutic compound for topical formulation ranges from about 0.01 mg / ml to about 100 mg / ml. Typically, the concentration of therapeutic compound for topical formulations ranges from about 0.1 mg / ml to about 10 mg / ml. Solid dispersions of therapeutic compounds as well as dissolved preparations can be used. Thus, the correct concentration is subject to moderate empirical manipulation and optimizes the therapeutic response. About 1000 mg of therapeutic compound per 100 grams of vehicle is useful in the treatment of skin lesions and provides a 1.0% weight / weight (w / w) formulation. Suitable vehicles include oil-in-water or water-in-oil emulsions using mineral oil, petratam, and the like, as well as gels such as hydrogels.

  Alternative topical formulations include shampoo preparations, oral pastes, and mouthwash preparations. The concentration of the therapeutic compound is typically as mentioned for the topical formulation.

  The therapeutic compound is optionally administered topically by use of a transdermal therapeutic system (see Barry, Darmatological Formulations, Marcel Dkker, 1983, p. 181 and references cited therein). Although such topical delivery systems have been largely designed for transdermal administration of low molecular weight drugs, by definition they are capable of percutaneous delivery. These can be readily adapted for administration of the therapeutic compounds of the present invention by appropriate selection of a rate controlling porous membrane.

  Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the therapeutic compound together with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary depending on the requirements of the particular compound, but are typically nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), non-toxic proteins such as serum albumin, sorbitan esters, oleic acid, lecithin Amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

  Solid or liquid unit dosage forms can be prepared for oral administration. In order to prepare solid compositions such as tablets, the therapeutic compounds are combined with conventional ingredients as pharmaceutical diluents or carriers such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, Formulated by mixing with lactose, gum arabic, methylcellulose and functionally similar materials. Capsules are prepared by mixing the therapeutic compound with an inert pharmaceutical diluent and filling the mixture into suitably sized hard gelatin capsules. Soft gelatin capsules are prepared by mechanical encapsulation of a therapeutic compound slurry with an acceptable vegetable oil, light liquid petrolatum or other inert oil.

  Fluid unit dosage forms for oral administration such as syrups, elixirs, and suspensions can be prepared. The water-soluble form can be dissolved in an aqueous vehicle together with sugar, aromatic flavoring agents and preservatives to form a syrup. An elixir can be prepared by using a hydroalcoholic (eg, ethanol) vehicle with a suitable sweetener, such as sugar and saccharin, with an aromatic flavor. Suspensions can be prepared in aqueous vehicles with the aid of suspending agents such as gum arabic, gum tragacanth, methylcellulose and the like.

  Appropriate formulations for parenteral use will be apparent to those of ordinary skill in the art. Typically, the therapeutic compound is prepared in an aqueous solution at a concentration of about 0.01 to about 100 mg / ml (discussed below). More typically, the concentration is from about 0.1 to about 10 mg / ml. The formulations are sterile and suitable for a variety of parenteral routes including intradermal, intra-articular, intramuscular, intravascular and subcutaneous.

  In addition to the therapeutic compound, the composition is a pharmaceutically acceptable, non-toxic carrier or diluent, depending on the desired formulation, to form a pharmaceutical composition for animal or human administration. Including those commonly used vehicles. The diluent is selected so that it does not unduly affect the biological activity of the combination. Examples of such diluents that are particularly useful for injectable formulations are water, various saline solutions, Ringer's solution, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may include additives such as other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.

  In addition, excipients can be included in the formulation. Examples include cosolvents, surfactants, oils, wetting agents, softeners, preservatives, stabilizers and antioxidants. Any pharmaceutically acceptable buffer may be used, such as Tris or phosphate buffer. Effective amounts of diluents, additives and excipients are those amounts effective to obtain a pharmaceutically acceptable formulation in terms of solubility, biological activity and the like.

  The term `` unit dosage form '' includes a predetermined amount of active material calculated to produce the desired pharmaceutical effect associated with the pharmaceutical diluent, carrier or vehicle for which each unit is required. A physically distinguishable unit suitable as a unit dose for human subjects and animals. The specifications for the unit dosage forms of this invention include (a) the unique characteristics of the active material, and the specific effects to be achieved and (b) such active material for use in humans and animals. Defined and dependent on the limitations inherent in the matching technology.

  Examples of unit dosage forms are tablets, capsules, pills, powder pockets, wafers, suppositories, granules, cachets, teaspoonful, tablespoonful, droppersful, ampoules, vials, metered release Aerosols of any of the foregoing, separated into a plurality, and other forms as described herein.

  Thus, the compositions of the present invention can be formulated with vehicles for conventional, pharmaceutically acceptable topical, oral or parenteral administration. The formulation may also contain small amounts of adjuvants such as buffers and preservatives to maintain isotonicity, physiology, and pH stability. Preparation, formulation and means of administration are known in the art. See generally Remington's Pharmaceutical Science 15th ed., Mack Publishing Co., Easton, PA /. (1980).

  Slow or extended release delivery systems, systems using liposomes, and polymeric delivery systems containing any number of biopolymers (biological based systems) are utilized with the compositions described herein for therapeutic compounds. Can provide a continuous or long-term source. Such slow release systems are applicable to formulations for topical, ophthalmic, oral, and parenteral use.

  The therapeutic agents of the invention are usually delivered or administered by topical or transdermal patches to treat hypertrophic or keloid scarring. Alternatively, oral administration is used. Additionally, the agent can be delivered parenterally or by direct injection of skin lesions. Parenteral treatment is typically intradermal or intravenous.

  A preferred way to practice the invention is to apply the therapeutic compound directly to the scar or lesion in a cream or oil-based carrier. Typically, the concentration of therapeutic compound in the cream or oil is 0.1-10%. Alternatively, aerosol can be used locally. These compounds can also be administered orally. The thiazolidinedione compound pioglitazone is an example of a thiazolidinedione that can be used in this manner.

  In general, the route of administration is topical (including the eye, scalp, and mucosa), oral, or parenteral. Topical administration is preferred for the treatment of skin lesions and includes scalp, corneal lesions, and mucosal lesions, where such direct application is practical. Mouthwash and oral paste formulations can be advantageous for mucosal lesions, such as oral lesions and leukoplakia. Oral administration is a preferred variant for the treatment of skin lesions and other lesions mentioned above, where direct topical application is not practical and it is the preferred route for other applications.

  Intradermal injection is a preferred variant for one or only a few (eg 2-6) lesions. Usually, the compound is delivered in an aqueous solution of about 0.1-10 mg / ml.

  An effective amount of the therapeutic compound is used in the treatment. The dose of the compound used in the present invention will vary depending on the compound and the condition being treated. The age, weight, and clinical condition of the recipient patient and the experience and judgment of the clinician or practitioner administering the treatment are among the factors influencing the dose selected. Other factors include the route of administration, the patient, the patient's medical history, the severity of the disease process and the potential of the particular compound. The dose should be sufficient to alleviate the symptoms or signs of the disease to be treated without producing unacceptable toxicity to the patient.

  Broadly, the dosing schedule is about 0.1 to about 600 mg twice a day. More typically, a single dose will give about 1-200 mg of compound twice a day. A convenient oral dose for adult patients is 10 mg twice a day. The dosage range for topical treatment is about 0.1% to about 10% (weight / volume) in cream, gel or oil, applied twice a day. Topical administration for intradermal administration is about 0.1-10 mg per injection per site.

  Typically, a dose is administered at least once a day until a therapeutic result is achieved. Preferably, the administration is twice a day, although more or less frequent administration can be recommended by the clinician. Once a therapeutic outcome is achieved, the drug can be reduced or stopped. Occasionally, side effects justify discontinuing treatment. In general, an effective amount of a compound is one that provides subjective removal of symptoms or an objective identifiable improvement noted by a clinician or other qualified observer.

  The foregoing serves in principle for illustrative purposes. It will be readily apparent to those skilled in the art that operating conditions, materials, procedural steps, and other parameters of the systems described herein may be further modified or substituted in various ways without departing from the spirit and scope of the invention. For example, although the present invention has been described for a human patient as a normal recipient, veterinary applications are also contemplated. Thus, the invention is not limited by the preceding description, but rather is by the appended claims. All cited references are hereby incorporated by reference.

Example 1
Myofibroblast count after treatment with pioglitazone Human dermal fibroblasts were cultured on sterile coverslips and fibroblast medium, ie (1.0 ml L-glutamine (200 mM; Gibco), 1.0 ml). Non-essential amino acids, NEAA (100 ×; Gibco), 1.0 ml penicillin / streptomycin 8100 × Gibco), 10 ml newborn calf serum, NBCS (Gibco), 87 ml Dubecco's modified Eagle medium, DMEM (Gibco)) for 24 hours. . At confluence, the medium was supplemented with myofibroblast differentiation medium, ie 1 ng / ml TGF-beta1 (Sigma), 1.0 mL L-glutamine (200 mM; Gibco), 1.0 mL non-essential amino acids, NEAA (100x; Gibco ), 1.0 mL penicillin / streptomycin (100 × Gibco), 1 mL ITS (insulin, transferrin, selenium (100 × Gibco)). Myofibroblast differentiation was allowed to proceed for 3 days in the presence or absence of various concentrations of pioglitazone.

  Fibroblast numbers were determined by immunohistochemistry according to the following protocol. Cells were washed with phosphate buffered saline (PBS), then fixed with 3% paraformaldehyde for 5 minutes and then quenched with 50 mM ammonium chloride. The cells were then washed and permeabilized with 0.2% Triton X-100 in PBS. All antibodies were diluted in 0.2% fish skin gelatin (FGS) (Sgima). Prior to antibody detection, non-specific binding was blocked using 0.2% FGS in PBS. Mouse anti-human alpha smooth muscle actin (Sigma) was diluted 1: 100 and incubated with cells for 45 minutes and then washed. Rabbit anti-mouse immunoglobulin horseradish peroxidase secondary antibody was incubated for 30 minutes. For visualization, Fast-DAB (Sigma) was used according to the manufacturer's instructions. Cells were counterstained with hemotoxylin.

  Images were captured using a Nikon Eclipse E-1000 microscope coupled to a Lucia image analysis system. Hemotoxylin stained nuclei were counted by software using size exclusion parameters to exclude small and large debris. The accuracy was determined in software using a binary overlay of counted objects and testing whether any non-nucleus count occurred or no nuclei were present. The software was found to be accurate over 99%. DAB positive cells were counted manually on the captured images. Counts were expressed as% positive cells / total cells.

  Experimental conditions were optimized over the number of experiments and typically produced about 10% of the total number of cells during the control treatment. Fibroblasts were easily identifiable as normal fibers compared to cells. Treatment with pioglitazone reduced the number of fibroblasts present at the end of the experimental period. This was clearly visible where the control slide exhibited easily identifiable fibroblasts compared to increasing doses of pioglitazone (FIG. 1).

The data presented in FIG. 1 indicated that pioglitazone reduced the number of myofibroblasts as a percentage of the total cell count in a dose response manner. Results are expressed as% myofibroblasts and ± SEM of total cells. * P <0.05, p <0.005

Example 2
Morphology of myofibroblasts treated with pioglitazone in vivo The morphology of fibroblasts and myofibroblasts after treatment with pioglitazone was evaluated by phase contrast microscopy. See FIG. Cells labeled with arrows appear to contract and thus undergo apoptosis. This cell appears to have the remaining prominent cytoskeleton when compared to fibroblasts marked with an arrowhead. The prominent cytoskeleton suggests that the cells labeled with arrows are myofibroblasts (x200). These data suggest that there was abundant cell death at higher concentrations of pioglitazone and that the dying cells were myofibroblasts and that this was mediated by apoptosis.

Example 3
Cell contraction and cytoplasmic blebbing in pioglitazone-treated myofibroblasts Human skin myofibroblasts were cultured and differentiated into myofibroblasts in myofibroblast differentiation medium supplemented with 1 ng / ml TGF-beta. In this experiment, differentiation occurred in the presence of 1 μM pioglitazone. The results are shown in FIG. As described in Example 1, as a percentage of the total cell number, pioglitazone reduced the number of myofibroblasts. Certain types of alpha smooth muscle actin positive cells that were present showed typical myofibroblast morphology and properties suggesting apoptosis. This included membrane blebbing and cytoskeletal disruption.

A: 0 μM, B: 0.1 μM, C: 1 μM, D: 10 μM pioglitazone in fibroblast differentiation medium supplemented with 1 ng / ml TGF-beta1. Arrows indicate examples of DAB positive myofibroblasts. A: fibroblasts (x100) cultured in differentiation medium supplemented with 1 ng / ml TGF-beta1 in the absence of pioglitazone, B: differentiated fibroblasts (x100) cultured with pioglitazone (10 μM) , C: high magnification magnified view (x200), differentiated fibroblasts showing cells considered to be myofibroblasts based on abundant and expanded cytoskeletal filaments (arrows), D: pioglitazone treatment ( 10 μM) High-magnification enlarged view of differentiated fibroblasts (x200). A: Myofibroblasts from cultures not exposed to pioglitazone treatment showing typical size, expanded morphology and predominant myofibrils (arrowheads). B: Pioglitazone-treated culture showing α-smooth muscle positive cells (Oya), clearly contracted and rounded compared to the surrounding normal fibroblasts. There is also evidence of membrane blebbing, suggesting apoptosis rather than necrotic death. C and D: Pioglitazone treated cultures. Myofibroblasts labeled with large arrows show strong but still scattered cytoplasmic staining.

Claims (19)

Of activators of PPARγ in the manufacture of a medicament for the treatment of conditions affecting the skin, characterized by impaired fibroblast or myofibroblast function, excess matrix production, nodular fasciitis or dupuitlan contracture use. Use according to claim 1, wherein the condition comprises keloid or hypertrophic scarring. 2. Use according to claim 1, wherein the condition is scleroderma. Use according to claim 1, wherein the condition is nodular fasciitis or Dupuytren's contracture. Use according to any of claims 1 to 4, wherein the activator is thiazolidinedione. Use according to claim 5, wherein the activator is 5-phenyl-thiazolidinedione. 6. Use according to claim 5, wherein the activator is 5-benzyl-thiazolidinedione. Use according to claim 5, wherein the activator is 5- (naphthyl or benzothienyl) thiazolidinedione. Use according to any of claims 1 to 4, wherein the activator is pioglitazone, rosiglitazone, siglitazone, troglitazone, isaglitazone, darglitazone or englitazone. Use according to claim 9, wherein the activator is pioglitazone. Use according to any of claims 1 to 4, wherein the activator is 4-phenyloxyethyl-5-methyl-2-phenyloxazole. Use according to any of claims 1 to 4, wherein the activator is 15-deoxy-delta-12,14-prostaglandin J2. The activator has the structure I

Wherein R ₁ is a 5-membered carbocyclic acid or 1 or 2 nitrogen, oxygen or sulfur atoms and 1 or 2 nitrogen atoms, optionally substituted with a (C ₁ -C ₃ ) alkyl group A heteroaryl group and R ₂ is 2- (5-methyl-2-phenyloxazol-4-yl) ethyl or 2- (2-pyridylmethylaminoethyl)]
The use according to any of claims 1 to 4, which is a benzophenone derivative. Use according to any of claims 1 to 4, wherein the activator is structure II.

[Wherein R ₁ is C ₁ -C ₅ alkyl, R ₂ is a halo-substituted pyridyl group, and R ₃ is a phenyl group]
Use of which is a sulfonamide derivative. 15. Use according to any of claims 1 to 14, wherein the medicament is adapted for topical administration. 15. Use according to any of claims 1 to 14, wherein the medicament is adapted for intralesional or subcutaneous administration. Use according to any of claims 1 to 16, wherein the medicament comprises 1 to 400 mg of activator. 18. Use according to any of claims 1 to 17, wherein the condition is hypertrophic scarring. 19. Use according to claim 18, wherein the activator is selected from thiazolidinedione, troglitazone, pioglitazone, rosiglitazone and thioglitazone.

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